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Journal of Bacteriology, March 2004, p. 1811-1817, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1811-1817.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus

Department of Chemistry, University of Utah, Salt Lake City, Utah 84112

Received 19 June 2003/ Accepted 1 October 2003

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His₆-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni²⁺ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70°C and pH 6.5. NADPH, flavin mononucleotide, and Mg²⁺ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K_m, 64 µM; specific activity, 0.476 µmol mg^-1 min^-1; and k_cat, 1.6 s^-1.

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